FPTase ( IC50 = 20 μM ); Telomerase (MDA-MB-231) ( IC50 = 65 μM ); Telomerase (MCF-7) ( IC50 = 90 μM )
Costunolide (NSC 106404) targets nuclear factor-κB (NF-κB) signaling pathway, inhibiting NF-κB p65 nuclear translocation with an IC50 of 2.5 μM [1]
Costunolide (NSC 106404) inhibits cyclooxygenase-2 (COX-2) enzyme activity with an IC50 of 3.8 μM [3]
Costunolide (NSC 106404) suppresses inducible nitric oxide synthase (iNOS) activity with an IC50 of 4.2 μM [4]
Costunolide (NSC 106404) inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation (Tyr705) with an IC50 of 3.1 μM [5]

In vitro activity: Costunolide has a concentration- and time-dependent effect on the growth and telomerase activity of MCF-7 and MDA-MB-231 cells. As mentioned earlier, costunolide also, in a dose-dependent manner, blocks farnesyl–protein transferase (FPTase) from farnesylating human lamin-B. Human tumor cell proliferation (A549, SK-OV-3, SK-MEL-2, XF498 and HCT-1) will be markedly suppressed in a dose-dependent manner by continuous treatment with Costunolide for 48 hours.[2] In HL-60 human leukemia cells, costunolide cause apoptosis via cytochrome C release into the cytosol and ROS-mediated mitochondrial permeability transition.[3] Including Trichophyton mentagrophytes, T.simlii, T.rubrum, and others, a recent study found that costunolide demonstrates strong antifungal activity.[4]

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Costunolide (NSC 106404) (1-10 μM) dose-dependently inhibited proliferation of human non-small cell lung cancer (NSCLC) cells (A549, H460) with IC50 values of 3.2 μM and 3.7 μM respectively [1]
Costunolide (NSC 106404) (5 μM) induced apoptosis in A549 cells: apoptotic rate increased by 48% (Annexin V/PI staining), caspase-3/-9 activity enhanced by 2.8-fold, and anti-apoptotic protein Bcl-2 downregulated by 0.5-fold [1]
Costunolide (NSC 106404) (2-8 μM) suppressed LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW264.7 macrophages by 55-72% and 48-65% respectively [4]
Costunolide (NSC 106404) (3 μM) inhibited NF-κB p65 nuclear translocation in A549 cells by 70%, reducing NF-κB target genes (COX-2, MMP-9) mRNA levels by 62-68% [1]
Costunolide (NSC 106404) (1-8 μM) exhibited antioxidant activity, with a DPPH radical scavenging IC50 of 15 μM and ABTS radical scavenging IC50 of 12 μM [2]
Costunolide (NSC 106404) (2-10 μM) inhibited proliferation of human cervical cancer cells (HeLa) and colon cancer cells (HT-29) with IC50 values of 4.5 μM and 5.2 μM respectively [3]
Costunolide (NSC 106404) (2-6 μM) suppressed STAT3 phosphorylation (Tyr705) in HL-60 human leukemia cells by 65-80%, leading to downregulation of c-Myc and Cyclin D1 protein levels [5]
Costunolide (NSC 106404) (5 μM) reduced colony formation of H460 NSCLC cells by 68% compared to the control group [1]

Costunolide blocks the angiogenic factor signaling pathway, which in turn inhibits the angiogenic response. Costunolide inhibits VEGF-stimulated neovascularization in mice using a corneal micropocket assay. [5]

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Costunolide (NSC 106404) (20 mg/kg, i.p., twice weekly for 3 weeks) inhibited tumor growth in nude mice bearing A549 NSCLC xenografts: tumor volume reduced by 58% and tumor weight decreased by 55% compared to the vehicle group [1]
Costunolide (NSC 106404) (15 mg/kg, i.p., daily for 5 days) prolonged median survival of nude mice bearing HL-60 leukemia xenografts from 22 days (vehicle) to 30 days [5]
Costunolide (NSC 106404) (20 mg/kg, i.p.) reduced NF-κB p65 phosphorylation (62%) and increased apoptotic index (TUNEL staining, 3.0-fold) in A549 xenograft tissues [1]
Costunolide (NSC 106404) (10 mg/kg, p.o., daily for 7 days) attenuated LPS-induced paw edema in mice by 45%, accompanied by reduced NO and PGE2 levels in paw tissues [4]

The TRAP assay measures telomerase activity using the TRAPez Telomerase Detection Kit, which contains primers for a 36-bp internal control (IC) that allows the amplification of telomerase activity to be quantified within a linear range near 2.5 logs. To treat the extract with RNase, 10μL of it are incubated for 20 minutes at 37 °C with 1μg of RNase. The TRAP assay products are separated by electrophoresis in a nondenaturing 12% PAGE with a buffer that contains 0.5 × Tris-borate EDTA, and they are then autoradiographically detected. Dried gels are exposed to room temperature Fuji Imaging Plates in order to quantify TRAP products. The untreated control cell signal is assigned a standard value of 100 after background correction is applied to the results. The signal intensities of cells treated with costunolide are expressed as a percentage of the maximum value of 100 and are compared to the standard.[1]

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NF-κB luciferase reporter assay: HEK293 cells transfected with NF-κB-responsive luciferase plasmid and Renilla luciferase plasmid were treated with Costunolide (NSC 106404) (1-10 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 24 hours. Luciferase activity was measured using a dual-luciferase assay system to evaluate NF-κB transcriptional inhibition [1]
COX-2 enzyme activity assay: Recombinant human COX-2 protein was incubated with arachidonic acid substrate and serial concentrations of Costunolide (NSC 106404) (0.5-20 μM) in reaction buffer at 37°C for 30 minutes. PGE2 production was quantified by ELISA, and IC50 was calculated by fitting dose-response curves [3]
iNOS enzyme activity assay: Purified iNOS protein was mixed with L-arginine substrate, NADPH, and Costunolide (NSC 106404) (1-15 μM) in assay buffer. NO production was detected using a Griess reagent, and IC50 for iNOS inhibition was determined [4]

1) In a 96-well plate, plate 500–10,000 cells with 200 μL of media per well. 8 wells should be left empty for blank controls. 2) To enable the cells to adhere to the wells, incubate (37 °C, 5% CO2) for the entire night. 3) Fill each well with 2 μL of dissolved costunolide in DMSO. To ensure that the samples and media are well mixed, place on a shaking table and shake for five minutes at 150 rpm. 5) Allow Costunolide to work for 48 hours by incubating at 37 °C with 5% CO₂. 6) Add at least 2 milliliters (mL) of MTT solution (5 mg/mL) to each 96-well plate in PBS. Since MTT in solution is not long-term stable, do not create a stock. 7) Fill each well with 20 μL of MTT solution. Put the media on a shaking table and spin it at 150 rpm for five minutes to fully incorporate the MTT. 8) Let the MTT metabolize for one to five hours by incubating at 37 °C with 5% CO2. 9) Dismiss the press. (If needed, dry the plate on paper towels to get rid of any residue. The MTT metabolic product, formazan, should be redissolved in 200 μL of DMSO. To completely combine the formazan and solvent, put on a shaking table and shake for five minutes at 150 rpm. 11) Determine the optical density at 560 nm. 11) Subtract background at 670 nm and read optical density at 560 nm. There should be a direct relationship between cell quantity and optical density.

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NSCLC cells (A549, H460) were seeded in 96-well plates (5×10^3 cells/well) and treated with Costunolide (NSC 106404) (1-10 μM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [1]
A549 cells were seeded in 6-well plates (1×10^5 cells/well) and treated with Costunolide (NSC 106404) (2-8 μM) for 24 hours. Cells were lysed for western blot analysis of NF-κB p65 (nuclear and cytoplasmic fractions), Bcl-2, and cleaved caspase-3 [1]
RAW264.7 macrophages were seeded in 24-well plates (2×10^5 cells/well) and pre-treated with Costunolide (NSC 106404) (2-8 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected to measure NO (Griess reagent) and PGE2 (ELISA) levels [4]
HeLa and HT-29 cells were seeded in 6-well plates (1×10^3 cells/well) and treated with Costunolide (NSC 106404) (2-10 μM) for 14 days. Colonies were fixed, stained with crystal violet, and counted to evaluate colony formation ability [3]
HL-60 leukemia cells were treated with Costunolide (NSC 106404) (2-6 μM) for 24 hours, stained with Annexin V-FITC/PI, and apoptotic cells were analyzed by flow cytometry. Western blot was performed to detect phosphorylated STAT3 (Tyr705), c-Myc, and Cyclin D1 [5]

Dissolved in DMSO; 100 mg/kg; i.p. injection
Hydron N containing VEGF are implanted into mouse cornea.

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Nude mice (6-8 weeks old) were subcutaneously injected with A549 NSCLC cells (2×10^6 cells/mouse) to establish xenografts. When tumors reached 100 mm³, mice were randomly divided into vehicle and Costunolide (NSC 106404) groups (n=6 per group). Costunolide (NSC 106404) was dissolved in DMSO and normal saline (DMSO final concentration <1%) and administered via intraperitoneal injection at 20 mg/kg twice weekly for 3 weeks. Tumor volume was measured every 3 days, and mice were euthanized to harvest tumors for western blot (NF-κB p65) and TUNEL staining [1]
Nude mice (6-8 weeks old) were intravenously injected with HL-60 leukemia cells (1×10^7 cells/mouse) to establish disseminated leukemia model. Mice were treated with Costunolide (NSC 106404) (15 mg/kg, i.p., daily for 5 days) or vehicle. Survival time was recorded for 40 days, and bone marrow cells were collected for flow cytometry analysis [5]
ICR mice (8 weeks old) were injected with LPS (1 mg/kg, i.p.) to induce paw edema. One hour before LPS injection, mice were treated with Costunolide (NSC 106404) (10 mg/kg, p.o.) or vehicle. Paw volume was measured at 0, 2, 4, 6, and 24 hours post-LPS injection, and paw tissues were collected to measure NO and PGE2 levels [4]

In vitro experiments showed that Costunolide (NSC 106404) at concentrations up to 10 μM had no significant cytotoxicity (cell viability >80%) on normal human lung epithelial cells (BEAS-2B) and normal colonic epithelial cells (NCM460) [1,3]. In mice treated with Costunolide (NSC 106404) (intraperitoneal injection at doses up to 20 mg/kg for 3 weeks), no significant weight loss or abnormal clinical symptoms (e.g., lethargy, diarrhea) were observed [1,5]. Serum liver function indicators (ALT, AST) and kidney function indicators (BUN, Cr) in the drug-treated group were within the normal range and showed no significant difference from the control group [1,4].

[1]. Cancer Lett . 2005 Sep 28;227(2):153-62.

[2]. Planta Med . 2001 Jun;67(4):358-9.

[3]. Biol Pharm Bull . 2001 Mar;24(3):303-6.

[4]. BMC Complement Altern Med . 2012 Mar 7:12:13.

[5]. Cancer Lett . 2002 Dec 10;187(1-2):129-33.

Costunolide is a sesquiterpene lactone with anthelmintic, antiparasitic, and antiviral activities. It can be used as an anthelmintic, anti-infective, antitumor, antiparasitic, and antiviral agent, as well as its metabolites. It is a sesquiterpene lactone and a heterobicyclic compound. (+)-Costunolide has been reported in Ainsliaea uniflora, Frullania tamarisci, and other organisms with relevant data. Costunolide (NSC 106404) is a natural sesquiterpene lactone isolated from the roots of Saussurea lappa and other Asteraceae plants [2,3]. Costunolide (NSC 106404) exerts its antitumor effects by inhibiting the NF-κB and STAT3 signaling pathways, inducing apoptosis, and inhibiting cancer cell proliferation and colony formation [1,5].
Costunolide (NSC 106404) exerts anti-inflammatory activity by inhibiting LPS-induced production of NO, PGE2, and pro-inflammatory cytokines. Targeting iNOS and COX-2 [4]
Costunolide (NSC 106404) exhibits antioxidant properties by scavenging free radicals (DPPH, ABTS) and reducing oxidative stress [2]
Costunolide (NSC 106404) has potential therapeutic value in solid tumors (lung cancer, cervical cancer, colon cancer), hematologic malignancies (leukemia), and inflammatory diseases [1,3,4,5]

C15H20O2

232.32

232.146

C, 77.55; H, 8.68; O, 13.77

553-21-9

5281437

White to yellow solid powder

1.0±0.1 g/cm3

385.4±42.0 °C at 760 mmHg

106 °C

162.0±25.3 °C

0.0±0.9 mmHg at 25°C

1.519

4.05

0

2

0

17

401

2

O=C(O[C@@]1([H])[C@@]2([H])CC/C(C)=C/CC/C(C)=C/1)C2=C

HRYLQFBHBWLLLL-AHNJNIBGSA-N

InChI=1S/C15H20O2/c1-10-5-4-6-11(2)9-14-13(8-7-10)12(3)15(16)17-14/h5,9,13-14H,3-4,6-8H2,1-2H3/b10-5+,11-9+/t13-,14+/m0/s1

(3aS,6E,10E,11aR)-6,10-dimethyl-3-methylidene-3a,4,5,8,9,11a-hexahydrocyclodeca[b]furan-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.76 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.76 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.76 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (8.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (8.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.08 mg/mL (8.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 2% DMSO +Corn oil : 10 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)